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Selective antibiotics

General questions

Can multiple selective antibiotics be used to stably select the same cell line?

Indeed, any selective antibiotics can be used together at the same time and in any combination.

Why isn’t it recommended to add selective antibiotics during initial cell culture?

We recommend not adding selective antibiotics until the cells have been passaged twice as cells are fragile during the initial culture procedure and the antibiotics may reduce cell viability. The resistance marker has to be expressed enough for the cells to acquire the resistance to these selective antibiotics.

Do you have a protocol on how to deactivate InvivoGen’s selective antibiotics?

For deactivation of all our selective antibiotics (ZeocinTM, Hygromycin, G418, Phleomycin, Puromycin and Blasticidin), we would recommend adding to the growth medium:
- 5% NaOH (Sodium hydroxide)
- 0.1% NaClO (Sodium hypochlorite)
You should then leave to react for 5 – 10 minutes and this should effectively deactivate all antibiotics in media.

Do you have data on the half-life of InvivoGen’s selective antibiotics in cell culture media?

Unfortunately we do not have data on the half-life of our selective antibiotics in cell culture medium. In house we prefer to add the selective antibiotics extemporaneously however if necessary you can supplement the medium with the antibiotic of your choice and keep at 4°C for no more than 1 week.

What is the preferred storage method for InvivoGen’s selective antibiotics?

All of our selection antibiotics are shipped at room temperature. Upon receipt, they should be stored at 4°C or -20°C. Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened they remain stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and they can be kept at 4°C for 3 months providing they are kept in sterile conditions.

Hygromycin B Gold

In the TDS it is stated that less Hygromycin B Gold (50-100 µg/ml) can be used in low salt LB, however we only have premixed high salt LB in our lab, therefore what concentration of Hygromycin B Gold should be used for selection?

We recommend to use Hygromycin B Gold at a concentration of 100 – 150 µg/ml for high salt LB conditions

What is the preferred storage method for Hygromycin B Gold?

Hygromycin B Gold is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label. Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

What are the selection conditions in mammalian cells?

The working concentrations of Hygromycin B Gold for mammalian cell lines vary from 50 to 200 μg/ml; in a few cases, up to 500 μg/ml. In a starting experiment we recommend to determine the optimal concentration of Hygromycin B Gold required to kill your host cell line. In general, Hygromycin B Gold is used at a concentration of 200 μg/ml, a 500-fold dilution from the stock solution. For more detailed information, please refer to the technical data sheet for this product.

What are the selection conditions in E. coli?

Hygromycin-resistant transformants are selected in low-salt LB agar medium (yeast extract 5g/l, tryptone 10 g/l, NaCl 5 g/l, agar 15 g/l, pH 8) supplemented with 50 to 100 μg/ml of Hygromycin B Gold.
Plates containing Hygromycin B Gold are stable for 1 month when stored at 4 °C.

Zeocin™

Is the Zeocin™ you provide hydrochloric or sulfate?

The Zeocin™ we supply is hydrochloric.

Is Zeocin™ photosensitive?

No, Zeocin™ is not photosensitive.

Is Zeocin™ pH sensitive?

Zeocin™ will undergo irreversible denaturation at high and low pH [6 - 8] or in the presence of a weak oxidant.

In what can we dilute the Zeocin™ solution to make intermediate working solutions?

Further dilutions to create an intermediate working solution are performed in sterile water.

What is the concentration of HEPES buffer used to reconstitute Zeocin™?

The concentration of HEPES buffer used to reconstitute Zeocin™ is 5 g/L

What is the preferred storage method for Zeocin™?

Zeocin™ is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once Zeocin™ solution opened, it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

What are the selection conditions in mammalian cells?

The working concentration of Zeocin™ for mammalian cell lines varies from 50 to 400 μg/ml, in a few cases can be as low as 20 μg/ml or as high as 1000 μg/ml. In a starting experiment we recommend to determine the optimal concentration of Zeocin™ required to kill your host cell line. For more detailed information, please refer to the technical data sheet for this product.

What are the selection conditions in E.coli?

Zeocin-resistant transformants are selected in Low Salt LB agar medium (yeast extract 5 g/l, Tryptone 10 g/l, NaCl 5 g/l, Agar 15 g/l, pH 7.5) supplemented with 25 μg/ml of Zeocin™.
Note: Do not use an E. coli recipient strain that contains the Tn5 transposable element (i.e. MC1066). Tn5 encodes a bleomycin-resistance gene that will confer resistance to Zeocin™.

Phleomycin

What is the preferred storage method for Phleomycin?

Phleomycin is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once Phleomycin solution opened, it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions

What are the selection conditions in mammalian cells?

The working concentration of phleomycin for mammalian cell lines varies from 5 to 50 μg/ml. In a starting experiment we recommend to determine the optimal concentration of phleomycin required to kill your host cell line. For more detailed information, please refer to the technical data sheet for this product.

What are the selection conditions in in E.coli?

The cells of the common E. coli recipient strains (i.e. HB101, DH5α, MC1061) transformed by vectors carrying bleomycin resistant genes, such as Sh ble and Tn5, are resistant to phleomycin.
Phleomycin-resistant transformants are selected in Low Salt LB agar medium (yeast extract 5g/l, Tryptone 10g/l, NaCl 5g/l, Agar 15 g/l, pH 7.5) supplemented with 5 μg/ml of phleomycin.

G418

Why does one of my bottles of G418 solidify when stored at -20°C, while another remains completely liquid and translucent?

We actually observe the same phenomenon internally also. The explanation lies in the fact that even below 0°C, the freezing reaction of the solution does not start spontaneously: it needs a disturbance to start. This disturbance can be a vibration, a shock, etc...
Please note however that as soon as solidification has occurred in the liquid, it behaves like a chain reaction and propagates quickly throughout the available volume of solution.
According to our scientists, we often observe differences in solidification between several tubes of G418 which is explained by a small shock to a tube and not the others.

What is the preferred storage method for G418?

G418 is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

What are the selection conditions in mammalian cells?

The working concentration of G418 Sulfate for selection and maintenance of mammalian cell lines transfected with the neo gene varies with a multitude of factors including cell type. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line by treating the cells with several concentrations, ranging from 100 μg/ml to 1 mg/ml.
Please note that G418 is normally used at a concentration of 400 μg/ml.

My plasmid has the Neo gene. Can I use G418 to select E. coli transformants?

No. G418 is used to select mammalian cells expressing the Neo gene. The appropriate antibiotic for selection in E. coli expressing the Neo gene is Kanamycin.

Puromycin

What is the best way to resuspend Puromycin?

We recommend to resuspend Puromycin in HEPES buffer (5 mM, pH 7.3)

We have noticed a crystalline precipitate of Puromycin when thawing a vial left at -20°C, is this normal?

During storage a crystalline precipitate of puromycin may form. If this occurs, heat the product at 37 °C for 30 minutes until the crystalline precipitate disappears. The formation of a crystalline precipitate does not affect the activity of the product.

What is the preferred storage method for Puromycin?

Puromycin is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

What are the selection conditions in mammalian cells?

The working concentrations of puromycin for mammalian cell lines range from 1 to 10 μg/ml. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene. For more detailed information, please refer to the technical data sheet for this product.

What are the selection conditions in E.coli?

Puromycin is poorly active on E.coli. However, puromycin-resistant transformants can be selected in LB agar medium supplemented with 100-125 μg/ml of puromycin [pH adjusted to pH 7.5 - 8].

Blasticidin

What is the preferred storage method for Blasticidin?

Blasticidin is shipped at room temperature.
Upon receipt, it should be stored at 4°C or -20°C.
Avoid repeated freeze-thaw cycles.
The expiry date is specified on the product label.
Once Blasticidin solution opened, it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.
Regarding Blasticidin powder, the stock solution must be at a concentration of 10 mg/ml and aliquots can be kept 1 year at -20°C (below 10 mg/ml we cannot guarantee the stability of the product). Again, avoid repeated freeze-thaw cycles.

What are the selection conditions in mammalian cells?

The working concentration of Blasticidin for mammalian cell lines varies from 1 to 10 μg/ml, in a few cases up to 30 μg/ml. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. For more detailed information, please refer to the technical data sheet for this product.

What are the selection conditions in E.coli?

E.coli is poorly sensitive to blasticidin, but transformants resistant to blasticidin can be selected on low salt LB agar medium (pH 8) supplemented with 100 μg/ml blasticidin. High pH enhances the activity of blasticidin.

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